The proteasome is thought to be a central component of cellular protein degradation machinery, but little is known about the mechanisms by which it is regulated. In preliminary data presented in this proposal, we report evidence that calmodulin is a major regulator of the proteasome. Proteasomes appear to have three distinct binding sites for calmodulin, located on the subunit Rpn11 and on the proteasome-associated proteins Hul5 and Ubp6. Remarkably, all three of these "receptors" for calmodulin act enzymatically on proteasome-bound ubiquitin chains. We have characterized specific amino acid substitutions in the calmodulin-binding sites each of these proteins. None of these mutations is detectably hypomorphic for proteasome function. However, when the mutations are crossed into the canonical proteasome hypomorph rpn4, which cannot adjust its proteasome levels to compensate for functional defects, they result in a major recovery of proteasome function. Thus, calmodulin apparently binds directly to the proteasome to inhibit its activity. Remarkably, the calmodulin binding sites in the proteasome are strictly conserved from yeast to humans, suggesting that the regulatory mechanism described here is universal. We propose a detailed analysis of proteasome regulation by calmodulin, based on a combination of biochemistry and genetics. We will investigate what aspect of proteasome activity is altered by calmodulin, prominent candidates including the enzymatic activities of Rpn11, Hul5, and Ubp6. We will also determine whether calcium is involved in the regulation, and whether regulators that function downstream of calmodulin, such as calmodulin-dependent kinases and calcineurin, are involved in the pathway. The studies will elucidate a surprising new signaling pathway that couples two major regulatory systems of eukaryotes. Based on the multiplicity of calmodulin binding sites on the proteasome, and the key roles of each of the proteasome's calmodulin receptor proteins, this already appears to be a highly intricate regulatory pathway. PUBLIC HEALTH RELEVANCE: The proteasome is very significant for human health;proteasome inhibitors have proven to be quite effective in clinical use against several cancers and more recently, proteasomes have been linked to many other diseases, with increasingly detailed understanding. This proposal describes the discovery of what appears to be a major new pathway for regulating the proteasome via a central cellular regulator, calmodulin. A better understanding of this new pathway may potentially shed new light on a variety of disease mechanisms.